The long-range goal of our work is to understand how protein structure governs function, to determine how proteins with altered structures acquire new properties. Such changes can be responsible for disease states. The objective of this proposal is to examine structure, stability, and folding mechanism in E. coli trp repressor. We will use molecular genetics and biochemistry to develop altered repressors, and compare their stabilities, folding rates, and structures with those of wild-type. The work will be organized around four specific aims: three kinds of mutagenesis of the trp R gene, and a biochemical approach to alter the Trp R protein. Aim 1. A selection for temperature-sensitive mutations will be used to isolate mutants bearing heat- and cold-sensitive repressors following chemical mutagenesis of the trp R gene. The stability of each protein to thermal and chemical denaturation will be determined using circular dichroism spectropolarimetry (CD), and folding rates will be determined. Crystallization and NMR studies will be used to determine the structural consequences of mutation. The goal of this work is to gain an understanding of which amino acid residues of a protein contribute to the stability of the folded state, and how they do so. Aim 2. Key amino acid residues in the hydrophobic core of wild-type trp repressor will be identified from the x-ray crystal structure. Initially, one of these will be mutated to all 19 other residues by codon randomization using oligonucleotide-directed mutagenesis. Characterization of the repressors purified from an initial group of mutants will be similar to that described for ts mutants above. These experiments should extend our understanding of the nature of the hydrophobic core and its role in folding and stability. Aim 3. A specific amino acid replacement will be made at a predetermined site within one helix of the repressor. The dynamics of this helix, as well as folding rates and overall protein stability, are all affected by changes at this site; an explanation for these effects will be tested by the mutation described. The stability and folding rates of the purified mutant protein will be measured by CD, and its structure and dynamics will be analyzed by NMR. These experiments test how the stabilization of a single helix contributes to the overall stability of the protein, and examine the detailed mechanisms for that stabilization. Aim 4. The protein will be dissected by enzymatic proteolysis and chemical cleavage to generate fragments corresponding to or containing folding units. The stabilities and folding rates of fragmentsalone and when recombined will be determined by CD, and structural characterization by NMR or x-ray will be attempted. These experiments are aimed at determining the definitions of, and relationship between, folding units and domains of the protein, and may provide a simple model for certain steps of the folding pathway.